Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately before the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein.
Conventional diagnostic evaluation of periodontal disease has relied on measuring clinical parameters, such as probing depth, attachment level, and plaque accumulation, and on measuring the height of the alveolar bone using radiographs. One shortcoming of these conventional tests is that they only define the status of the periodontium at the time of examination. In the past 10 to 15 years, studies have shown that clinical parameters of periodontal disease are poor predictors of when and at which sites patients that would experience active disease (Haffajee, A. D., et al., 1993). In addition, measurement of alveolar bone loss using intraoral radiographs is of limited value because it provides only a historical record of past disease and cannot be used to determine when the loss of crestal bone occurred.
Due to these limitations, researchers have investigated alternative methods for evaluating patients with periodontal disease (Lamster, I. B., et al., 1993).
Previously, analysis of the host inflammatory and immune responses in periodontal disease has been performed on gingival crevicular fluid (GCF). Among the host-derived mediators shown to identify patients at risk for active periodontal disease is the lysosomal enzyme, .beta.-glucuronidase (Lamster, I. B., et al., 1988; Lamster, I. B., et al., 1995), which is a marker of primary granule release from neutrophils into the gingival crevicular fluid. Lamster and coworkers previously found that elevated levels of .beta.-glucuronidase in gingival crevicular fluid were diagnostic for existing periodontal disease, and correlated with the likelihood of future disease progression (Lamster, I. B., et al., 1994; Lamster, I. B., et al., 1995). Existing tests for .beta.-glucuronidase in gingival crevicular fluid involve collecting fluid from within the gingival crevice using such special devices as methylcellulose strips or microsyringes. The levels of .beta.-glucuronidase are measured in the fluid by adding a substrate for .beta.-glucuronidase which in the presence of the enzyme produces a product that is detectable. Unfortunately, this method is labor-intensive and requires the services of highly-trained personnel, such as a dentist or dental hygienist. Therefore, although measurements of .beta.-glucuronidase levels in gingival crevicular fluid have been shown to be a better indicator of human periodontal disease than previous conventional evaluations, the limitations of this method make its widespread use unlikely. Currently no method exists that overcomes these disadvantages and provides a simple, reliable test for diagnosing periodontal disease which could be utilized on a widespread basis.
Although one might consider using samples from other body fluids such as saliva that are easy to collect, distinct drawbacks for biochemical evaluation of periodontal disease by analysis of saliva exist (Lamster, I. B. and Grbic, J. T., 1995).
More specfiically, based on the current state of the art as described in the scientific literature, it would have been expected to be difficult, if possible, to detect elevated levels of .beta.-glucuronidase in saliva as a means of diagnosing periodontal disease (Ding, Y., et al., 1994; Lundy, F. T. and Lamey, P-J., 1995). Thus, detection of markers in general is more difficult in saliva than in gingival crevicular fluid because constituents of saliva are derived from many sources, including the major and minor salivary glands and from gingival crevicular fluid. (Lamster, I. B. and Grbic, J. T., 1995). Further, the relatively large volumes of fluid involved when dealing with saliva would be expected to have both a masking and a diluting effect on any potentially important marker derived from gingival crevicular fluid (Lamster, I. B. and Grbic, J. T., 1995), thus making it difficult to detect the marker.
Moreover, even if detection of the enzyme in saliva was possible, one would have expected that it would be difficult to distinguish between healthy and diseased subjects. Although previous studies have proposed diagnostic tests in saliva for periodontal disease using markers such as IgG, IgA and collagenase, none has ever mentioned the use of .beta.-glucuronidase. (Lamster, I. B. and Grbic, J. T., 1995). In fact, some experts have argued that elevated levels of markers in saliva would not be detectable until after disease had become widespread, thereby limiting the importance of such an approach for diagnositic purposes (Mandel, I., 1991). Furthermore, even if .beta.-glucuronidase was detectable, one could reasonably have expected that the dilution of the marker by the much larger volume of saliva would have made it impossible to distinguish between healthy and diseased subjects.
Based on the foregoing, one skilled in the art would not have expected that a test for measuring the concentration of .beta.-glucuronidase in saliva would be useful for the diagnosis of periodontal disease. Unexpectedly, a saliva test based on measuring levels of .beta.-glucuronidase has not only proven useful, it has proven to be a better discriminator of periodontitis than analysis of gingival crevicular fluid or clinical parameters.